A cone, which is a female flower formed on a female plant of hop (Humulus lupulus) that is a dioecious plant, is an important raw material used for brewing beer. The cone of hop contains bitter acid, terpenoid, polyphenol, and the like, and is deeply associated with the flavor of beers. Since only the cone is utilized as the raw material, only the female plant is used for cone production. Also, as an unpollinated cone is a good quality raw material, male plans are not only unneeded at cone-producing districts but male plants become a cause of poor quality and is hence not cultivated intentionally.
Meanwhile, the male plant is utilized for breeding. The breeding of hop is practiced by cultivating, propagating and evaluating plant individuals derived from seeds obtained by crossbreeding the pollen of a male flower formed on a male plant with the female flower of a female plant, and screening for a desirable individual. The utilized male plant cannot form a female flower thereon, and thus the cone as the raw material cannot be evaluated. Accordingly, a male plant is utilized on the presumption of genetic abilities thereof based on cone evaluation of sister individuals thereof or cone evaluation of offsprings produced with other female plants. However, hop is a vegetative propagation crop and has high genetic heterozygosis which makes it difficult to presume such ability, and thus an efficient breeding strategy on the cone evaluation is in demand. Further, the male plant is required to be maintained only to be utilized for the breeding.
To solve these problems and practice the breeding efficiently, a strategy is conceived that the male plant is not utilized, that is, a male flower is intentionally induced to develop on a female plant to crossbreed female plants with each other. Up to date, it is documented that a male flower was formed on a hop female plant by spraying 500 ppm α (2-chlorophenylthio)propionic acid, sodium salt, wherein an involvement with auxin, a plant hormone, was examined (Non Patent Literature 1). However, this document merely describes that the male flower was formed and it is not clear whether the pollen formation, fertility, and the like, were achieved therein. Regarding the formation of an male flower on a hop female plant, the use of such an auxin-related substance was the only single report documented half a century ago, and α (2-chlorophenylthio)propionic acid has not been utilized for the breeding of hop ever since.
In the dioecious plants whose sexuality is genetically determined, the XY type is the most common but the ZW type is also known, and the sex determination is roughly divided into the species in which the male sex determination factor is presumably present in the Y chromosome even in the XY type and the species in which the sex is determined by the ratio of the X chromosome to the autosome (Non Patent Literature 2), thus suggesting a diverse genetic background in the sex determination. It is reported that the sexuality of hop is determined by the ratio of the X chromosome to the autosome (Non Patent Literature 3). Non Patent Literature 4 describes that morphic sex chromosomes were found in asparagus and hop and thus the sex is determined by the presence of sex gene on the sex chromosomes. On the other hand, this literature indicates, in reference with the following examples, that plant hormones are involved with the sex determination in many plants having no such property. Auxin and ethylene promote the feminization in the Cucurbitaceae (cucumber), pineapple, papaya and date, and gibberellin promotes the masculinization in the Cucurbitaceae, mulberry (mulberry belonging to the Moraceae) and oil palm, and cytokinin induces a hermaphrodite flower in the male grape. Further, the literature refers that the actions rendered by these plant hormones include many exceptions. For example, auxin promotes the formation of Pseudotsuga male buds, and ethylene promotes the masculinization of Chinese chestnut, and female flowers of locust beans gum and date palm contain a higher level of gibberellin than male flowers thereof and the addition of gibberellin promotes the feminization in corn and Chinese chestnut, or the like. Regarding such a confusing ununified incomprehensible situation, Non Patent Literature 4 points out that such a situation might be caused by a mismatch between the physiological plant hormone level and the chemical concentration externally administered. Also, this literature describes that, even in the Cucurbitaceae which has been well studied, the part of a plant, environmental factors (i.e., light intensity, nutrient conditions, temperature, day length conditions) influence the changes in sex. As described above, Non Patent Literature 4 suggests that the sex determination of hop is not associated with plant hormones but depends on morphic sex chromosomes. Further, the literature includes examples illustrating the conflicting effects that different plant hormones promote the feminization or the masculinization depending on the plant species or concentrations in a case of the plant with which plant hormones are associated.
Plant hormones are, in many cases, involved with the sex determination and sexual differentiation of plants which is documented in auxin, gibberellin, cytokinin, ethylene, and the like, and there is an opinion that there is no plant hormone which works commonly on higher plants (Non Patent Literature 2). Furthermore, in recent years, it is suggested that in corn, which is a dioecious flower, the pistil is degenerated due to jasmonic acid, which is one of plant hormones, and transformed to the male flower (Non Patent Literature 5). Thus, in the sex determinations in the plants, mechanisms differ due to the various plant hormones and so it is considered that each has independently evolved. The study on the sex conversion in plant species associated with ethylene has been progressed using the Cucurbitaceae crops. The Cucurbitaceae crops are dioecious plants, and female flowers and male flowers are respectively formed on a genetically identical single plant by physiological factors. In the Cucurbitaceae crops, a case of inducing female flowers by ethylene treatment is known, and in cucumber it is suggested that the stamen development is inhibited by ethylene and an female flower is formed thereon (Non Patent Literature 6). Further, it is reported that a hermaphrodite flower is formed when a female plant of Momordica dioica plant belonging to the Cucurbitaceae, a dioecious plant, is treated with silver nitrate, which is an ethylene inhibitor (Non Patent Literature 7). Furthermore, it is reported that in Cannabis sativa belonging to the Cannabis, a dioecious plant, when silver nitrate or STS (silver thiosulphate anionic complex), an ethylene inhibitor, is applied to a growing shoot tip, which becomes black, an intersex flower, an incomplete male flower with few stamens or a complete male flower, together with female flowers, were formed on the main branch and lateral branches which grow thereafter (Non Patent Literature 8).